Angiogenesis is a process wherein a new capillary is generated from an existing microvessel. Normal angiogenesis occurs in embryonic development, tissue regeneration and wound treatment, and luteinization—a periodic change in the female reproductive system—and it proceeds under strict control (Folkman J. et al., Int. Rev. Exp. Pathol., 16, pp. 207-248, 1976).
Vascular endothelial cells grow very slowly in adults and are rarely divided compared with other types of cells. The process of angiogenesis progresses with decomposition of the basal membrane of blood vessels by protease due to the stimulation of angiogenesis-stimulating factor, migration and proliferation of vascular endothelial cells, and tubular formation due to differentiation of vascular endothelial cells, which results in reconstruction of blood vessels to form a new capillary.
Several diseases are caused by failure of self-regulation of angiogenesis and abnormal growth of blood vessels. Angiogenesis-related diseases occurring in pathological conditions include angioma, angiofibroma, vascular malformations, cardiovascular diseases such as atherosclerosis, vascular adhesion, scleredema and ophthalmic diseases such as keratoplastic angiogenesis, neovascular glaucoma, diabetic retinopathy, neovascular corneal diseases, spot degenerations, pterygium, retinal degeneration, retrolental fibroplasia and granular conjunctivitis. Chronic inflammatory diseases such as arthritis, skin diseases such as psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis and acne, Alzheimer's and obesity are also related to angiogenesis. Furthermore, growth and metastasis of cancer are necessarily dependent on angiogenesis (D'Amato R. J. et al., Ophthalmology, 102(9), pp. 1261-1262, 1995; Arbiser J. L., J. Am. Acad. Dermatol., 34(3), pp. 486-497, 1996; O'Brien K. D. et al. Circulation, 93(4), pp. 672-682, 1996; Hanahan D. et al., Cell, 86, pp. 353-364, 1996).
Especially in cancer, angiogenesis plays an important role in the growth and metastasis of cancer cells. Tumors are provided with nutrients and oxygen by new blood vessels, which infiltrate the tumors and give cancer cells an opportunity to enter into blood circulation to aid metastasis of cancer (Folkman and Tyler, Cancer Invasion and Metastasis, Biological Mechanism and Therapy (S. B. Day, ed.) Raven Press, New York, pp. 94-103, 1977; Polverini P. J., Crit. Rev. Oral. Biol. Med. 6(3), pp. 230-247, 1995). A principal reason that chemotherapies or immunotherapies for cancer patients scarcely contribute to increasing the survival rates of patients is metastasis of cancer.
Arthritis, a typical inflammatory disease, is primarily induced by an autoimmune disorder. As the disease progresses, chronic inflammation in synovial bursa between the joints induces angiogenesis and destroys cartilage. That is, synovial cells and vascular endothelial cells proliferate with the aid of inflammation-inducing cytokine, and as angiogenesis progresses, joint pannus, which is connective tissue layer generated in cartilage, is formed to destroy the cushioning cartilage (Koch A. E. et al., Arthritis. Rheum., 29, pp. 471-479, 1986; Stupack D. G. et al., Braz, J. Med. Biol. Res., 32(5), pp. 578-581, 1999; Koch A. E., Arthritis Rheum., 41(6), pp. 951-962, 1998).
Ophthalmic diseases, which make millions of people blind every year, result from angiogenesis (Jeffrey M. I. et al., J. Clin. Invest., 103, pp. 1231-1236, 1999). For example, macular degeneration occurring in the aged, diabetic retinopathy, retinopathy of a precocious child, neovascular glaucoma and neovascular corneal diseases are caused by angiogenesis (Adamis A. P. et al., Angiogenesis, 3, pp. 9-14, 1999). Among the diseases, diabetic retinopathy is a complication of diabetes which results in blindness due to the invasion of retinal capillaries into the vitreous body.
Psoriasis, characterized by red spot and scaled skin, is a chronic proliferative disease occurring in skin that is difficult to treat, and involves pain and deformation. While in a normal state keratin cells proliferate once a month, in psoriasis patients they proliferate once a week. For such rapid proliferation, profuse blood must be provided, which forces active angiogenesis (Folkman J., J. Invest. Dermatol., 59, pp. 40-48, 1972).
Anti-angiogenic agents can be applied to treat various angiogenic diseases. Since anti-angiogenic agents are generally administered to patients for a long time, those of low toxicity and which are orally administered are ideal. Thus, there is a great need to develop a novel anti-angiogenic agent with low toxicity.
For a long time, the present inventors have studied macrolide antibiotics having a 24-membered lactone ring, macrolactine derivatives including macrolactin A and their pharmacological mechanisms of action. Macrolactin compounds are produced from unclassified ocean bacteria, actinomyces and Bacillus strains (Willam Fenical et al., J. Am. Chem. Soc., 111, pp. 7519-7524, 1989; Ik Dong Yoo et al., J. Microbiol. Biotechnol., 7, pp. 429-434, 1997; Gabriella Molinari et al., Antimicrob. Agents Chemother., 50, pp. 1701-1709, 2006), and 23 macrolactin compounds have been identified. Prior studies on the pharmacological activities of the macrolactin compounds are as follows.
MA, isolated for the first time in 1989, suppresses the proliferation of murine melanoma cancer cell and Herpes simplex virus, and protects T-lymphoblast damaged by HIV (Wiliam Fenical et al., J. Am. Chem. Soc., 111, pp. 7519-7524, 1989). Furthermore, it suppresses squalene synthase (Sung Won Choi et al., Can. J. Microbiol., 49, pp. 663-668, 2003), protects brain cells damaged by glutamate (Ik Dong Yoo et al., J. Microbiol. Biotechnol., 7, pp. 429-434, 1997) and has an antibacterial activity against various pathogens including super bacteria (Gabriella Molinari et al., Antimicrob. Agents Chemother., 50, pp. 1701-1709, 2006). In addition, the present inventors confirmed that MA and its derivatives inhibit the formation of inflammatory mediators induced by lipopolysaccharide (LPS) and filed a patent application (PCT/KR2010/003239).
The present inventors confirmed that MA, MMA and SMA were produced from Bacillus polyfermenticus KJS-2 (KCCM10769P, hereinafter, “BP2”) fermentation or culture medium and identified their structures. Besides the three macrolactin compounds, the present inventors also confirmed by LC/Mass that macrolactin B, macrolactin C, macrolactin D, macrolactin E, macrolactin F, macrolactin G, macrolactin H, macrolactin I, macrolactin J, macrolactin K, macrolactin L, macrolactin M, macrolactin N, isomacrolactinic acid and macrolactinic acid were produced from the BP2 fermentation or culture medium crude extract (Korean Patent No. 10-0895908). However, the present inventors did not confirm the production of macrolactin O, macrolactin P, macrolactin Q, macrolactin R or macrolactin S from BP2.
Macrolactin compounds have various pharmacological activities for their various structures. However, studies about the anti-angiogenesis of macrolactin compounds have never been reported until now. Hence, the present inventors fractionated the crude extract produced from the BP2 fermentation or culture medium including various macrolactin compounds using middle-pressure liquid chromatography (MPLC) and investigated anti-angiogenic activity of each fraction to find that the fractions containing macrolactin compounds suppress angiogenesis, and especially, the fractions containing MA, MMA and SMA strongly suppress angiogenesis. Accordingly, the present inventors highly purified each compound from the MPLC fractions containing the three macrolactin compounds (PCT/KR2010/003239) and investigated the anti-angiogenic activity of each purified macrolactin compound.